Calcium is a versatile intracellular signal messenger controlling numerous cellular functions. The Ca2+-signalling system operates in many different ways to regulate various cellular processes that function over a wide dynamic range. Calcium triggers exocytosis within microseconds and drives the gene transcription and proliferation in minutes to hours.
To understand these functions, fluorescence imaging with fluorescent probes such as Oregon Green 488 BAPTA-1 (OG1) and fura-2 have most often been used. However, use of these probes with one-photon microscopy requires excitation with short wavelength light (˜350-500 nm) that limits their application in tissue imaging owing to shallow penetration depth (<100 μm), photobleaching, photodamage, and cellular auto fluorescence.
Two-photon microscopy (TPM) overcomes these shortcomings. One-photon microscopy (OPM) employs one high-energy photon for excitation, whereas TPM employs two lower energy, near-infrared photons to produce an excited fluorescent substance. TPM has the advantages of localized excitation, increased penetration depth (>500 μm), lower cellular autofluorescence and self-absorption, as well as reduced photodamage and photobleaching, when compared to OPM. Thus, TPM allows imaging deep inside tissues for a long period of time without interference from artifacts of surface preparation that can extend >70 μm into the tissue slice interior.
However, most of the fluorescent probes presently used for TPM have small TP action cross sections (Φδ), demanding impractically high concentrations of probe and/or laser power. Furthermore, the fluorescence signals from membrane-bound probes can cause significant errors such as mistargeting because the fluorescence quantum yield is higher in the membrane than in the cytosol.
To the best of our knowledge, no study on two-photon dyes capable of selectively imaging calcium ions in real time has been reported. Therefore, there is a need to develop an efficient TP probe that can visualize the calcium waves deep inside the live tissue without photobleaching or mistargeting problems.